ES2580837T3 - Transformación de células somáticas en células madre neuronales reprogramadas inducidas (IRNSCS) - Google Patents
Transformación de células somáticas en células madre neuronales reprogramadas inducidas (IRNSCS) Download PDFInfo
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Abstract
Un método in vitro para producir Células Madre Neuronales (NCS), que comprende: a) proporcionar células somáticas de mamífero seleccionadas entre el grupo de fibroblastos, queratinocitos y adipocitos; y b) reprogramar dichas células somáticas en NSC por introducción, en las células somáticas, de al menos dos genes seleccionados entre el grupo de Bmi1 y Sox2 y cultivar las células somáticas en un medio que comprende factores de crecimiento seleccionados entre el grupo de FGF2, EGF y BDNF y un inhibidor de ROCK de molécula pequeña.
Description
GFP+ con un tamaño superior a 50 µm se cosecharon y se usaron adicionalmente para expansión. La mitad de las neuroesferas se expandieron usando el medio de expansión (N2B27 con FGF, 30 ng/ml de EGF 20 ng/ml de BDNF) con Fasudilo y la otra mitad sin Fasudilo. Día 15: Las neuroesferas cultivadas en el medio de expansión con Fasudilo tienen una morfología mejor y bordes claros y nítidos (una evidencia de neuroesferas bien
5 formadas, panel B); sin Fasudilo, las neuroesferas tienen bordes borrosos (panel A).
Figura 10: Caracterización de inmunocitoquímica de neuroesferas de irNSC para la expresión de los marcadores de NSC, Sox2 y Nestina. Día 15, las neuroesferas de irNSC expandidas con Fasudilo se sembraron en placas revestidas con PO/Lam y después de 48 h se tiñeron a la expresión de Sox2 y Nestina. Las neuroesferas de irNSC se unieron y las irNSC se propagaron desde las esferas. Las irNSC tienen una morfología de NSC habitual y eran positivas para Sox2 y Nestina. Panel A: Canales de fusión e individuales de DAPI, Sox2, Nestina; aumento 20x; Panel B: Canales de fusión de DAPI, Sox2, Nestina; aumento 10x.
Figura 11: Comparación de la estimulación con Fasudilo con respecto al compuesto 324 similar al Balanol para
15 generar neuroesferas de irNSC. Los fibroblastos humanos IMR90 se trataron con tripsina y se infectaron en un volumen pequeño con: Sox2, Bmi1, indicador de GFP de nestina usando el medio de inducción (Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con FGF, 20 ng/ml de EGF BDNF; 2 µg/ml de Heparina; 4 µg/ml de Polybrene) complementado con Fasudilo 10 µM (gráfico sombreado) o compuesto 324 similar al Balanol 2 µM (gráfico de color negro). Los fibroblastos se siembran en una placa de cultivo tisular normal a una concentración de 10000 -30000 células/cm2. Día 1: Cambiar el medio con medio de inducción recién preparado. Día 4: Se hizo el recuento de las neuroesferas GFP+ con un tamaño superior a 50 µm. El compuesto 324 pequeño similar al Balanol aumentaba la eficacia de la generación de neuroesferas aproximadamente dos veces (1,9) y tiene una mejor reproducibilidad (STDEV, n = 3).
25 Figura 12: El tratamiento previo de fibroblastos humanos con Ácido Valproico (VPA) aumenta el rendimiento de las neuroesferas de irNSC GFP+. Los fibroblastos humanos IMR90 se trataron previamente durante 48 horas con
o sin el inhibidor de HDAC, Ácido Valproico (ácido 2-propil-pentanoico, sal monosódica) (1 mM) antes de infección con: Sox2, Bmi1, indicador de GFP de nestina. Medio de inducción (Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con FGF, 20 ng/ml de BDNF de EGF; 2 µg/ml de Heparina; compuesto 324 similar al Balanol 2 µM). Día 7: Se hizo el recuento de las neuroesferas GFP+ con un tamaño superior a 50 µm (Panel A) y se indica el índice medio de irNSC GFP+ por neuroesfera (Panel B). Fotografías representativas de las neuroesferas de irNSC generadas con el tratamiento previo con VPA (Panel C). El tratamiento previo con VPA no influya de forma significativa en el número de neuroesferas el día 7; aunque el tratamiento con VPA aumentaba el número (2,1 veces) de irNSC GFP+ (STDEV, n = 3).
35 Figura 13: Definición de una combinación mínima de genes en combinación con Sox2 y Bmi1 para inducción eficaz de neuroesferas de irNSC. Los fibroblastos humanos IMR90 se trataron previamente durante 48 horas con VPA (1 mM) antes de infección con: Sox2, Bmi1, indicador de GFP de nestina más diferentes genes candidatos para dirigir su sinergia. Medio de inducción: Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con FGF, 20 ng/ml de BDNF de EGF; 2 µg/ml de Heparina y compuesto 324 similar al Balanol 2 µM. Cuantificación el Día 7 de neuroesferas de irNSC con un tamaño superior a 50 µm. Mash1, Emx2, Foxg1, Pax6 y Sox11 tienen sinergia con Bmi1 y Sox2 para generar neuroesferas de irNSC.
Figura 14: Generación de neuroesferas de irNSC a partir de fibroblastos dérmicos humanos de adulto (HDFa).
45 Los fibroblastos dérmicos humanos de adulto nos proporciona GIBCO (Número de Cat.: C-013-5C). Los fibroblastos dérmicos humanos de adulto se trataron con tripsina y se infectaron en un pequeño volumen con: Sox2, Bmi1, indicador de GFP de nestina usando el medio de inducción (Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con FGF, 20 ng/ml de BDNF de EGF; 2 µg/ml de Heparina) complementado con Fasudilo 10 µM. Día 8: Se detectan neuroesferas de irNSC (fotografías representativas con un aumento de 2,5 y 10X).
Figura 15: Expansión de neuroesferas de irNSC usando una combinación de Ácido Ascórbico, Hedgehog Sónica (Shh), Jagged1, DLL4 y FGF8 para obtener un cultivo de monocapa de las irNSC GFP+. Los fibroblastos humanos IMR90 se infectaron con: Sox2, Bmi1, Mash1 e indicador de GFP de nestina usando el medio de 55 inducción (Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con FGF, 20 ng/ml de BDNF de EGF; 2 µg/ml de Heparina; compuesto 324 similar al Balanol 2 µM). Día 7: Las neuroesferas con un tamaño superior a 50 µm se cosecharon y se expandieron adicionalmente con el medio de expansión (Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con FGF, 20 ng/ml de BDNF de EGF; 2 µg/ml de Heparina; compuesto 324 similar al Balanol 2 µM; Ácido Ascórbico 0,2 mM, 500 ng/ml de SHH (Hedgehog Sónica Humana Recombinante, Número de Catálogo: 1845SH), 100 ng/ml de FGF8 (Isoforma FGF8a Humana Recombinante, Número de Catálogo: 4745F8), 500 ng/ml de DLL4 (DLL4 Humana Recombinante, Número de Catálogo: 1506D4), 500 ng/ml de Jagged1 (Quimera de Fc de Jagged 1 Humana Recombinante, Número de Catálogo: 1277JG), medio acondicionado a 1/10 de las NSC derivadas de hESC cultivadas durante dos días en Kit de Proliferación de NS-A NeuroCult® (Humano, StemCells Technologies) con 65 FGF, 20 ng/ml de BDNF de EGF; 2 µg/ml de Heparina. Fotografías representativas para las neuroesferas de irNSC el día 14 expandidas con el medio de expansión informado anteriormente (Panel A). Las neuroesferas
8
Medio de Diferenciación: 20 ng/ml de N2B27 complementado con human BDNF (Roche), 2 µg/ml de Laminina (Invitrogen).
Fibroblastos humanos: fibroblastos de pulmón fetal IMR90 (Núm. de Lot. de la ATCC 580229699) o fibroblastos 5 dérmicos humanos de adulto (GIBCO, Número de Cat.: C-013-5C).
Lentivirus: Las partículas de lentivirus empaquetadas previamente, listas para usarse obtuvieron en Sigma (Lentivirus humano Sox2 Stemgent Reprogramming, n.º de Catálogo ST070012), Genecopeia (Partículas Lentivirales de Bmi1 de humano Lentifect, n.º de Catálogo LP-B0015-Lv105; Partículas Lentivirales de Sox11 Lentifect, n.º de Catálogo LP-MO425-LV105; Partículas Lentivirales de Mash1 Lentifect, n.º de Catálogo LP-Z0740-LV105; Partículas Lentivirales de Kpna1 humano Lentifect, n.º de Catálogo LP-U1286-Lv105; Partículas Lentivirales de NCam1 Lentifect, n.º de Catálogo LP-Z2645-Lv105) y SBI Systems Biosciences (Indicador de GFP de Nestina: Virus Indicador Transcripcional pGreenZeo™-hNestina, SR10035VA-1).
15 Títulos de 1,45* 10 5/µl de NestinaGFP, 4,3* 10 5/µl de BMI1, 1,07* 10 4/µl de Sox2, 3,2* 10 6/µl de Sox11, 4,7* 10 6/µl de Mash1, 3,3* 10 4/µl de NCam, 1,8* 10 5/µl de Kpna1.
Protocolos
1. Generación de las irNSC:
-200.000 IMR90 fibroblastos humanos infectados con los lentivirus para combinación de genes diferentes (multiplicidad de infección (M.O.I.) usada para cada lentivirus individual 30) y el lentivirus GFP de nestina indicador (M.O.I. usada 10) en un Eppendorf con 300 µl de medio de inducción con 4 µg/ml de Polybrene
25 (bromuro de hexadimetrina, Sigma).
- Incubar a temperatura ambiente durante 15 min.
-Sembrar los 300 µl en 1,7 ml de medio de inducción en un pocillo de una placa de 6 pocillos tratada con tejido
- Día 1, renovar los 2 ml de de medio de inducción/cada pocillo
-Día 3, cosechar las neuroesferas recogiendo con cuidado los 2 ml con las esferas flotantes 35 -Expandir las neuroesferas
2. Expansión de las neuroesferas:
-Recoger el medio con las neuroesferas flotantes en tubos de 15 ml de 3 pocillos de una placa de 6 pocillos -Dejar que las esferas sedimenten durante 10 min
45 -Retirar el sobrenadante con mucho cuidado (las células individuales no sedimentaran y se aspiran con el sobrenadante) -Volver a suspender las esferas en un medio de expansión a un volumen final de 4 ml -Sembrar en una placa B6 de adherencia ultra baja (Corning) -Incubar 2-3 días -Repetir el procedimiento de expansión cada 2-3 días hasta el día 14 desde la generación de las irNSC 55
3. Diferenciación de neuroesferas:
-Día 14 generación de las irNSC y después del procedimiento de expansión seleccionar las neuroesferas redondas en estereo microscopio con bordes claros y ricos en las irNSC GFP+
-Sembrar 40 esperasen una placa de 24 pocillos revestida previamente con poli-ornitina/laminina usando el medio de expansión con adición de Fasudilo 10 µM o compuesto 324 similar al Balanol 2 µM. -El día después renovar el medio de expansión sin Fasudilo / compuesto 324 similar al Balanol 2 µM.
65 -Incubar durante tres días
11
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Application Number | Priority Date | Filing Date | Title |
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EP10173455 | 2010-08-19 | ||
EP10173455 | 2010-08-19 | ||
PCT/EP2011/064051 WO2012022725A2 (en) | 2010-08-19 | 2011-08-16 | Conversion of somatic cells to induced reprogrammed neural stem cells (irnscs) |
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ES2580837T3 true ES2580837T3 (es) | 2016-08-29 |
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ES11743246.8T Active ES2580837T3 (es) | 2010-08-19 | 2011-08-16 | Transformación de células somáticas en células madre neuronales reprogramadas inducidas (IRNSCS) |
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US (2) | US9297025B2 (es) |
EP (1) | EP2606126B1 (es) |
JP (1) | JP5905006B2 (es) |
KR (2) | KR20160033798A (es) |
CN (1) | CN103068974B (es) |
BR (1) | BR112013003031A2 (es) |
CA (1) | CA2807226A1 (es) |
ES (1) | ES2580837T3 (es) |
HK (1) | HK1184492A1 (es) |
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Publication number | Priority date | Publication date | Assignee | Title |
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EP2606126B1 (en) | 2010-08-19 | 2016-05-18 | F.Hoffmann-La Roche Ag | Conversion of somatic cells to induced reprogrammed neural stem cells (irnscs) |
CN102604894B (zh) | 2012-02-29 | 2014-07-30 | 中国科学院广州生物医药与健康研究院 | 用于制备神经干细胞的培养基及其用途 |
PL223189B1 (pl) * | 2012-11-15 | 2016-10-31 | Celther Polska Spółka Z Ograniczoną Odpowiedzialnością | Komórka iNS oraz sposób reprogramowania komórek somatycznych za pomocą czynnika Sox2 oraz Sox2 i c-Myc do komórki iNS |
PE20151413A1 (es) * | 2012-11-21 | 2015-10-23 | Ptc Therapeutics Inc | Inhibidores de bmi-1 de pirimidina inversa sustituida |
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US5968829A (en) * | 1997-09-05 | 1999-10-19 | Cytotherapeutics, Inc. | Human CNS neural stem cells |
AU2002359390A1 (en) * | 2001-11-09 | 2003-05-19 | Artecel Sciences, Inc. | Endocrine pancreas differentiation of adipose tissue-derived stromal cells and uses thereof |
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EP2075254A1 (en) * | 2004-03-30 | 2009-07-01 | NsGene A/S | Therapeutic use of a growth factor, NsG33 |
GB0410011D0 (en) * | 2004-05-05 | 2004-06-09 | Novartis Forschungsstiftung | Neural cell differentiation method |
US8278104B2 (en) * | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
US20100021437A1 (en) * | 2008-04-07 | 2010-01-28 | The McLean Hospital Corporation Whitehead Institute for Biomedical Research | Neural stem cells derived from induced pluripotent stem cells |
WO2010018996A2 (ko) * | 2008-08-12 | 2010-02-18 | 연세대학교 산학협력단 | 인간 신경줄기세포 및 이를 이용한 중추 또는 말초 신경계 질환 및 손상 치료용 약학적 조성물 |
GB0902034D0 (en) * | 2009-02-06 | 2009-03-11 | Reneuron Ltd | Method |
EP2606126B1 (en) | 2010-08-19 | 2016-05-18 | F.Hoffmann-La Roche Ag | Conversion of somatic cells to induced reprogrammed neural stem cells (irnscs) |
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MX2013001587A (es) | 2013-03-21 |
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US20160369234A1 (en) | 2016-12-22 |
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HK1184492A1 (zh) | 2014-01-24 |
EP2606126A2 (en) | 2013-06-26 |
KR101788903B1 (ko) | 2017-10-23 |
RU2562111C2 (ru) | 2015-09-10 |
WO2012022725A3 (en) | 2012-04-12 |
CN103068974A (zh) | 2013-04-24 |
JP5905006B2 (ja) | 2016-04-20 |
RU2013110127A (ru) | 2014-09-27 |
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